PROJECT SUMMARY Aging is the dominant risk factor for fracture. Persons over 65 years of age account for 13% of the total US population, but represent > 50% of hospital admissions with a musculoskeletal injury. Fractures are the primary injury type among older patients, and increased age is a risk factor for impaired fracture healing. Delayed fracture healing in elderly patients results from a lower capacity for mesenchymal stem cell differentiation and impaired angiogenesis/vasculogenesis. Endothelial colony forming cells (ECFCs) are directly involved in angiogenesis and vasculogenesis. Thus, the decrease in number and/or function of ECFCs may be a major driver for failed fracture repair in elderly patients. In order to examine whether rescue of impaired angiogenesis may sufficiently enhance bone healing in the aged population, this proposal will dissect the contribution of angiogenesis factors, and evaluate the extent to which novel bone healing agents operate via stimulating endothelial cell (EC) function in a mouse model of bone healing with aging. We have shown in a rat critical sized defect (CSD) model, regeneration was accelerated when collagen scaffolds seeded with young ECFCs were implanted within the defect. We also show that the main megakaryocyte growth factor, thrombopoietin (TPO), can augment CSD healing in mice with concomitant increase in ECs. TPO can also heal CSDs in rats and pigs. Our recent data show that mRNA levels of Sirtuin-1 (Sirt1), an NAD+ class III histone deacetylase with anti-aging effects, were significantly higher in young CD45-CD31+ ECs as compared to old ECs. Additionally, Sirt1 mRNA significantly increased in the fracture regenerate of young mice compared to that of old mice. Further, new preliminary data showed that treatment with SRT1720 (sirt1 activator) improved bone healing. Based on these observations, we hypothesize that bone healing can be enhanced in aging by improvement of angiogenesis via direct implantation of ECFCs or through stimulation of ECs by TPO or SRT1720. In Aim 1, we will assess the use of young or old ECFCs as a bone regeneration therapy. In Aim 2, we will assess the therapeutic efficacy of TPO or SRT1720 on bone healing, and dissect the contribution of EC signaling to this response in aged mice. Successful accomplishment of these Aims will determine the degree to which the degeneration of ECs during aging is responsible for the age-related decline in the bone healing process. Additionally, using two distinct bone healing agents known to target ECs, we will identify age-specific differences in EC stimulation and how these differences influence the fracture repair process.